PROJECT 4: PROJECT SUMMARY Enhancing GBM Cytotoxic Therapy Through Inhibition of Key Autophagy Mediator ATG4B Glioblastoma (GBM) is the most common and malignant primary brain tumor. Despite treatment consisting of surgical removal, radiation and chemotherapy, most patients with GBM die within 14 to 16 months after diagnosis, underscoring the urgent need for new therapies to combat this deadly disease. Autophagy is a conserved catabolic process that maintains homeostasis by regulating the energy balance of the cell. Cancer cells use autophagy to remove damaged organelles and aggregated proteins, and to recycle nutrients in high demand to support tumor growth. Radiochemotherapy (RT-TMZ) is the front-line treatments against GBM, but also activate the autophagic response in tumor cells, thus protecting the cells from undergoing apoptosis. Inhibition of mTOR signaling is a common target in cancer therapy. However, mTOR inhibition also induces autophagy in cancer cells. Consequently, there is immense interest in inhibiting this protective mechanism while treating cancer. Non-specific autophagy inhibitors like chloroquine (CQ) and hydroxy-CQ (HCQ) are being investigated in a large number of clinical trials. However, the lack of specificity of these compounds is , associated with toxicity and may diminish its efficacy. We discovered that ATG4B, an enzyme that converts LC3 to LC3-I/II, which is required for autophagy process in cells, is phosphorylated at serine residue 383 (p-S383) in patient-derived glioma stem-like cells (glioma initiating cells or GICs). We also found that ATG4B S383 phosphorylation increases GIC autophagic activity, and that intratumoral expression of p-S383 ATG4B correlates with poor prognosis in GBM patients. In contrast, knockdown of ATG4B, or expression of a non- phosphorylatable ATG4B mutant transgene (S383A), inhibits GSC autophagic response and tumorigenicity when engrafted in the brains of athymic mice. Furthermore, pharmacologic inhibition of ATG4B, using a NCI compound NSC185058 that inhibits ATG4B enzymatic activity and GBM tumorigenicity, markedly enhanced RT inhibition on GBM tumor growth and increases the survival of animals with intracranially engrafted GIC. Additionally, NSC185058 also markedly enhanced inhibitory effects by a catalytic mTORC inhibitor AZD2014 on GICs. Based on these strong data and our established multiple PDX GBM models of distinct genetic + epigenetic profiles, in this project, we propose to determine the anti-GBM efficacy of ATG4B inhibitor NSC185058, as monotherapy and in combination with RT-TMZ; investigate the therapeutic potential of combining inhibition of ATG4B, mTOR and RT and develop NSC185058 for use in patients, and test the ATG4B inhibitor, both as a single agent and in combination with cytotoxic therapy, in a clinical trial for treating patients with recurrent GBM. This project has a strong molecular mechanistic foundation and associated therapeutic hypothesis that, if proven correct, has the potential for positive impact on the treatment of GBM, and perhaps other malignancies.